Mammalian reoviruses are highly virulent in newborn mice, producing injury to several organ systems, including the central nervous system and heart. Apoptotic cell death plays a major role in the pathogenesis of reovirus disease. Reovirus-induced apoptosis requires viral disassembly in cellular endosomes, intracellular signal transduction, and changes in the expression of cellular genes under the control of transcription factor NF-kB. Following reovirus infection, mice deficient in the p50 subunit of NF-kB display diminished apoptosis in the brain, yet enhanced apoptosis in the heart, as compared to wild-type animals. These features provide a tractable experimental system to investigate tissue-specific mechanisms of reovirus-induced apoptosis and pathogenesis. The central hypothesis of this proposal is that viral disassembly in the endocytic compartment stimulates the NF-kB signal transduction pathway, reprograms cellular gene expression, and either activates or suppresses the apoptotic machinery in a cell-type specific manner. Three integrated specific aims are proposed to test this hypothesis. In Specific Aim 1, mechanisms by which viral disassembly induces NF-kB activation and leads to apoptosis will be determined. These experiments focus on the membrane-penetration protein, ?1, which functions following disassembly to deliver the viral core into the cytoplasm and is genetically linked to strain-specific differences in apoptosis induction. Functional domains in ?1 that mediate apoptosis will be identified using mutant viruses and viral cores recoated with mutant forms of ?1. Apoptosis-defective ?1 mutants will be tested for the capacity to disrupt endosomes, injure mitochondria, and activate NF-kB. In Specific Aim 2, ?1-directed mechanisms of reovirus-induced NF-kB activation and apoptosis will be defined. Signaling intermediates that link ?1 with NF-kB during reovirus infection will be identified using genetically engineered cell lines and RNA interference. A novel pathway of NF-kB activation dependent on the IKK? catalytic and IKK? regulatory subunits of the multicomponent IkB kinase will be investigated. In Specific Aim 3, the role of tissue-specific NF-kB signaling pathways in reovirus pathogenesis will be elucidated. Mice lacking IKK?, IKK?, or NF-KB p50 in either the brain or heart will be infected with reovirus and assessed for apoptosis and disease. Tissue- specific p50-null mice will be used for microarray experiments to identify host genes under NF-kB control that are activated in response to reovirus infection in the brain and heart. Results of these experiments will provide insight into mechanisms by which viruses perturb cellular signaling pathways to cause cell death and disease. Information gained from these studies should foster development of new antiviral strategies designed to modulate apoptosis that may be applicable to a wide array of microbial pathogens. [unreadable] [unreadable] [unreadable]